Gel electrophoresis is a common procedure for the separation of biological molecules, such as DNA, RNA, and proteins. In gel electrophoresis, the molecules are separated into bands according to the rate at which an imposed electric field causes them to migrate through a filtering gel.
The basic apparatus used in this technique consists of a gel enclosed in a glass tube or sandwiched as a slab between glass or plastic plates. The gel has an open molecular network structure, defining pores which are saturated with an electrically conductive buffered solution of a salt. These pores through the gel are large enough to admit passage of the migrating molecules.
The gel is placed in contact with buffer solutions which make electrical contact between the gel and the cathode or anode of an electrical power supply. A sample containing the macromolecules and a tracking dye is placed on top of the gel. An electric potential is applied to the gel causing the sample macromolecules and tracking dye to migrate toward the bottom of the gel. The locations of the bands of separated macromolecules are then determined. By comparing the distance moved by particular bands in comparison to the tracking dye and macromolecules of known mobility, the mobility of sample macromolecules can be determined. Once the mobility of the sample macromolecules is determined, the size of the macromolecule can be calculated.
As more electrophoretic applications are used in quality control labs and forensic and clinical diagnoses, it is critical to be able to replicate all experimental conditions in multiple locations and labs, a very important variable being temperature. The application of an electrical field to a gel results in the generation of heat. In general, higher temperatures increase the molecular kinetics, which results in faster migration of macromolecules through the separating gel.
However, without temperature control, gels often exhibit uneven temperatures across the width of the gel resulting in "smile" distortions. Smile distortions occur when bands migrate faster in the middle of the gel than on the sides.
Often, even a small temperature differential between the front and rear plates of the gel, if not mitigated, can cause the resulting bands to slant front to back, depending on the thickness of the gel and the heat transfer properties of the cassette plates. This challenge is particularly acute in test runs where the molecular migration rates exhibit overly temperature sensitive characteristics, as in DNA sequencing. For such runs, even a slight temperature differential, e.g. of 0.1.degree. C. can cause the slanted bands to appear overlapping.
Additionally, overheating of the gel (&gt;60.degree. C.) can result in deleterious effects such as breakdown of the gel matrix resulting in poor resolution and band shape, alteration of the macromolecules including denaturation, alkylation or oxidation, and/or damage to the electrophoresis apparatus itself.
In DNA sequencing, electrophoresis is conducted at high voltage (1200-3000 volts, 55 watts) to maintain a gel temperature of 45.degree.-50.degree. C. for maximum resolution of the denatured DNA strands. The temperature is controlled by the amount of power applied to the gel. Gels run too cool (&lt;40.degree. C.) will have bands that are blurred, perhaps due to incomplete denaturation. Gels run too warm (&gt;60.degree. C.) will lose resolution, perhaps due to the breakdown of the polyacrylamide.
Precise temperature control is particularly critical in Single Stranded Conformational Polymorphism (SSCP) analysis of DNA, where bands are extremely close together. The relative temperature differential between the front and the back surfaces of the gel therefore can have a critical effect on the resolution of the DNA bands. For reliable, reproducible results, the gel temperature must be consistent for the duration of the run and in all areas of the gel, and variations of less than 0.1.degree. C. temperature differential between the front and the back surfaces of the gel are required.
Various means have been used to attempt to control the temperature of the gel during electrophoresis. These include applying active or passive heat sinks to one side of the gel, regulating power to the gel, employing an enclosed heat exchanger internal one of the buffer chambers, immersing the gels in a buffer-filled tank containing a heater/circulator, circulating the buffer through tubing immersed in an ice water bath, and circulating the buffer through an external metal heat exchanger.
These means are limited in their ability to provide compact apparatus for maintaining consistent and uniform thermal control across the area encompassing the front and back of the electrophoresis gels. The heat sinks exchange heat on only one side of the gel; the regulation of power to the gels cannot control regional hot spots and obviously limits the application of high wattage to the gels; the internal heat exchanger again exchanges heat on only one side of the gel and does not actively circulate buffer, resulting in vertical thermal gradients within the buffer chamber; immersing the gels in a heater tank is cumbersome, in that it requires a large volume of buffer and cannot cool the gels; and circulating the buffer through tubing immersed in an ice water bath is also cumbersome, and makes difficult fine control of temperature. Circulating the buffer through an external metal heat exchanger provides the most satisfactory temperature control. However, with the current electrophoresis systems, two pumps and heat exchangers would be required to assure uniformity of temperature and separation of the buffer fluids between the cathode and anode chambers. Further, with current electrophoresis systems, circulation of buffer within the chambers and across the gels is random and undirected, which may result in vertical and horizontal thermal gradients.
In view of the afore-mentioned deficiencies within the prior art, it is desirable to provide a compact system which requires only one heat exchanger, maintaining separation of the cathode and anode buffers while assuring consistency and uniformity of temperature across the front and the back of the electrophoresis gel.
It is also desirable for an electrophoresis system to provide the means for circulating a single buffer at any selected temperature (3.degree.-80.degree. C.) over multiple gels simultaneously in order to dissipate heat generated by the electrophoresis process and maintain a consistent thermal environment over a substantial portion of the gel surfaces using a single pump and heat exchanger.
It will be further desirable to allow the user to operate multiple temperature controlled electrophoresis units off of a single thermostatted circulating water bath due to the heat exchangers being located external to the bath.